Cells must identify the products of chromosome replication, termed sister chromatids, from S-phase until anaphase onset. However, the mechanism that first pairs sister chromatids together remains unknown. CTF7/ECO1 is the founding member of a cohesion establishment pathway and appears to couple sister chromatid pairing reactions to DNA replication. Sister pairing can also be induced outside of S-phase, suggesting that Ctf7p establishment activity is tightly regulated. Currently, Ctf7p remains the only essential establishment factor known. Recent findings confirm that Ctf7p functions during S-phase, is recruited to chromatin during S-phase and binds to numerous chromatin-associated replication factors. In Phase 1, we will use site-directed mutational analyses coupled to biochemical methodologies to identify which protein interactions are required for Ctf7p chromatin recruitment and how Ctf7p recruitment/activation is regulated. Genetic and molecular analyses will then be paired to high-resolution microscopy cohesion assays to test for the role of novel alleles in cell viability and in sister pairing reactions. In Phase 2, we will identify how Ctf7p pairs sister chromatids once recruited to chromatin. We will pursue results from our lab that Ctf7p associates with Pds5p, a cohesin regulator that is required to maintain sister pairing. Ctf7p-Pds5p binding is the only known link between the processes that establish and maintain cohesion. To address these issues, we will use biochemical and cell-cycle mapping strategies to test if Ctf7p-Pds5p binding is cell cycle specific, direct and essential. Both Ctf7p and Pds5p are post-translationally modified. Molecular and biochemical methodologies will be used to determine how Ctf7p-Pds5p establishment activity is regulated over the cell cycle, regulate binding and ultimately how Ctf7p- Pds5p affect cohesin dynamics on chromatin. [unreadable] [unreadable] [unreadable]